Abstract In the present study, Asp endoproteinase (AspN) was used to induce whey protein isolate (WPI) for preparing protein nanofibrils (PNFs) at 37 ℃. Transmission electron microscopy (TEM), atomic force microscope (AFM), tricine-SDS-PAGE, thioflavin T fluorescence spectroscopy, infrared spectroscopy and laser scattering were used for the characterization of PNFs and its formation process. The results showed that the PNFs was milky white in color, and peptides with a molecular weight of 5 kD were the building blocks for PNFs. The major secondary structure in PNFs was β-fold, which played a role in its stability, and the average particle size of PNFs was positively proportional to hydrolysis time. The enzymatic preparation of PNFs, overcoming the problem of browning, is expected to expand its application in food industry.